Hyperhomocysteinemia is detrimental to pregnancy in mice and is associated with preterm birth.

Elevated levels of homocysteine produce detrimental effects in humans but its role in preterm birth is not known. Here we used a mouse model of hyperhomocysteinemia to examine the relevance of homocysteine to preterm birth. The mouse carries a heterozygous deletion of cystathionine ß-synthase (Cbs(+/-)). Gestational period was monitored in wild type and Cbs(+/-) female mice. Mouse uterine and placental tissues, human primary trophoblast cells, and human myometrial and placental cell lines were used to determine the influence of homocysteine on expression of specific genes in vitro. The activity of BKCa channel in the myometrial cell line was monitored using the patch-clamp technique. We found that hyperhomocysteinemia had detrimental effects on pregnancy and induced preterm birth in mice. Homocysteine increased the expression of oxytocin receptor and Cox-2 as well as PGE2 production in uterus and placenta, and initiated premature uterine contraction. A Cox-2 inhibitor reversed these effects. Gpr109a, a receptor for niacin, induced Cox-2 in uterus. Homocysteine upregulated GPR109A and suppressed BKCa channel activity in human myometrial cells. Deletion of Gpr109a in Cbs(+/-) mice reversed premature birth. We conclude that hyperhomocysteinemia causes preterm birth in mice through upregulation of the Gpr109a/Cox-2/PGE2 axis and that pharmacological blockade of Gpr109a may have potential in prevention of preterm birth.

Microanatomy of secretory granule release from guinea pig tracheal goblet cells.

The microanatomy of mucin granule release from epithelial goblet cells has been investigated in guinea pig tracheae. Using a tannic acid arrest procedure, granule release under basal conditions and after high K+ or acetylcholine (ACh) application was arrested and a variety of granule fusion sites were identified in ultrathin sections and freeze-fracture replicas. Rather than there being subclasses of secretory cells containing either electron-lucent granules (indicative of mucin) or smaller electron-dense (serous) granules, the majority of secretory cells in both control and treated groups contained granules with an electrondense core surrounded by an electron-lucent region. Granule release sites were of three principal types: (1) simple exocytosis, where the membranes of single granules fused directly with the plasma membrane to give an "omega" profile; (2) compound exocytosis, where granule membranes, fused together intracellularly, were found in continuity with the plasma membrane; and (3) apocrine-like secretion, which involved the loss of the central apical mass of granules together with elements of the cell cytoplasm. In treated preparations, there was an increase in the number of cells exhibiting fusion sites; the percentage showing simple fusions fell from 82% to 59% (with ACh) and 57% (with KCl), whereas the percentage of cells exhibiting compound and apocrine-like secretion increased. Dense cores were frequently retained at the sites of fusion and, despite the expansive decondensation of mucin known to occur, there was also evidence of some retention of the electron-lucent material.

Ultrastructural characterization of tannic acid-arrested degranulation of permeabilized guinea pig eosinophils stimulated with GTP-gamma-S.

We have used ultrastructural techniques to investigate secretion in permeabilized eosinophils. As each exocytotic event is rapid we have used tannic acid incubation to trap the maximum number of fusion figures; tannic acid has been used previously in other secretory systems to arrest exocytosis at the cell surface whilst still allowing the preceding events to occur. Using this approach, in conjunction with ultrathin sectioning and cryoreplication, it is possible to demonstrate clear evidence of exocytosis in permeabilized eosinophils after stimulation by GTP-gamma-S. Large numbers of arrested fusion sites are found, including early fusion pedestals, visible in freeze-fracture replicas, having single narrow necked pores as small as 12 x 43 nm. Both individual and compound exocytoses are found, with retention of the secretory product, in particular the crystalline granule core, occurring at many sites. Large numbers of coated pits are also found in cells following extended tannic acid incubation, membrane coats even occurring on arrested granule membranes, suggesting a role in post-fusion membrane recovery. The accessibility of the cell interior and the large number of arrested fusion sites, particularly the presence of very early stages of exocytosis (evident as pedestals in freeze-fracture replicas), makes this a suitable preparation for the localization of key regulators of exocytosis at their sites of action. Although this approach, utilizing permeabilization coupled with tannic acid incubation is not without inherent problems-as with any electron microscopic technique care must be taken to understand the potential for artefacts-there are a number of advantages, particularly with regard to labeling studies, over techniques utilizing ultra rapid freezing.

Effect of neuromimetics upon the release of atrial natriuretic peptide granules: are multiple pathways involved in secretion?

The release of atrial natriuretic peptide (ANP) in response to the application of neurohumoral agonists (neuromimetics) is directly demonstrated and quantified at the cellular level, using an ultrastructural assay developed to quantify secretion. The assay uses an in situ tannic acid perfusion technique to arrest the exocytosis of atrial secretory granules in the anesthetized rat. The animal is perfused with the neuromimetic, and secretory granules, which retain the capacity to undergo exocytosis throughout the subsequent 30 min tannic acid perfusion, accumulate at the cell surface in a state of fusion with the plasma membrane. Quantification of arrested granules thus provides a measure of the rate of granule release and allows the responses to different agents to be assessed. The actions of three different agents were investigated: isoproterenol, phenylephrine, and acetylcholine. In previously published studies, investigations of the actions of these agents on ANP release has produced unclear and sometimes contradictory results. Using our ultrastructural assay, it was found that during the 30 min perfusion period neither isoprenaline nor phenylephrine caused a significant change in the rate of secretory granule release, whereas acetylcholine significantly decreased the rate of granule release. A new model of secretion is proposed to integrate these findings with previous results and help clarify the complex picture of atrial natriuretic peptide release.

Electrophysiological and ultrastructural events evoked by methacholine and intracellular photolysis of caged compounds in cultured ovine trachea submucosal gland cells.

Cultured ovine trachea submucosal gland cells release lysozyme in response to extracellular application of secretagogues, including the muscarinic receptor agonist methacholine (20 microM). Investigation of the ultrastructure has shown that these cells contain electron-dense cored granules, which differ from the intact tissue, but appear to be released in response to the application of methacholine and can be arrested during exocytosis by the application of tannic acid. The release process appears to be linked to electrophysiological events activated by methacholine. Extracellular application of methacholine and intracellular photorelease of Ca2+ from DM-nitrophen evoked similar events suggesting that a rise in intracellular Ca2+ may occur following muscarinic receptor activation. Measurements of the reversal potential and the inhibitory action of the chloride channel blocker niflumic acid (10 microM) indicated that Ca(2+)-activated Cl- channel activity underlies these events. Some of the cultured submucosal gland cells also responded similarly to intracellular photorelease of inositol 1,4,5-trisphosphate, suggesting a possible link between muscarinic receptor occupation by agonist, release of calcium from stores, and activation of Ca(2+)-activated Cl- current. Secretion of lysozyme, methacholine-activated currents and currents evoked by intracellular photorelease of Ca2+ were also attenuated by the potent bronchodilator Ro 31-6930 (1 microM). We conclude that Ca(2+)-activated Cl- conductances play an important role in secretory processes in cultured submucosal gland cells. This may have a bearing on both physiological control of secretory events and regulation of the nature of airway surface liquid. Ca(2+)-activated Cl- channels may offer a potential target site for novel therapeutic agents.

Identification of interleukin-2 in human peripheral blood eosinophils.

Interleukin-2 (IL-2) is an essential growth factor for T cells. Previous studies have shown that human peripheral eosinophils respond to IL-2 in chemotaxis and express the IL-2 receptor (CD25). In addition, eosinophils have been shown to transcribe messenger RNA for IL-2. The aim of the present study was to determine whether eosinophils translate mRNA for IL-2 and to determine the site of intracellular localization. By immunocytochemistry, an average of 9% of cells showed cytoplasmic staining for IL-2 in freshly isolated unstimulated blood eosinophils obtained from asthmatic subjects who were not receiving oral corticosteroid treatment (n = 5). Freshly isolated, disrupted, highly purified eosinophils (> 99%, by CD16- immunomagnetic selection) contained an average of 6 pg/10(6) cells of IL-2 measured by a specific enzyme linked immunosorbent assay (ELISA) (n = 7). Purified eosinophil incubated with serum-coated Sephadex beads showed an increase in the amount of intracellularly-retained IL-2 (26.2 +/- 7.2 pg/10(6) cells) with some evidence for release of this cytokine but only in three out of six eosinophil preparations (range 1.3-5.8 pg/10(6) cells). The intracellular localization of IL-2 was determined by fractionation of the cells on a linear (0-45%) Nycodenz gradient in sucrose buffer followed by detection of IL-2 in the fractions using an IL-2-specific ELISA and dot blotting. The majority of the IL-2 detected co-eluted with known eosinophil granule markers (i.e. major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil peroxidase (EPO) and beta-hexosaminidase) but small quantities were also detected in the cytosolic (lactate dehydrogenase-(LDH) associated) and membrane (CD9+) fractions. Immunogold labelling of intact eosinophils using an anti-IL-2 monoclonal antibody confirmed IL-2 immunoreactivity in association with the eosinophil crystalline granule cores. These data are consistent with the hypothesis that eosinophils synthesize, release and store IL-2 largely within cystalloid granules. This stored IL-2 may serve as a reservoir for rapid release of IL-2 in inflammatory reactions associated with eosinophilia.

Identification of messenger RNA for IL-4 in human eosinophils with granule localization and release of the translated product.

Human eosinophils are cytokine-producing cells that are prominent in IgE-dependent allergic tissue reactions. IL-4 promotes the development of the Th2-type phenotype in T cells and is an essential cofactor for IgE production by B cells. We detected mRNA for IL-4 by reverse transcription-PCR in blood eosinophils from atopic asthmatics. By specific ELISA, 108 +/- 20 pg of IL-4 protein/10(6) cells could be extracted from whole cells, and approximately 30% of the IL-4 was released after incubation with serum-coated particles. Using immunocytochemistry, eosinophils from atopic asthmatics and nonatopic controls showed IL-4 immunoreactivity using an anti-IL-4 mAb. IL-4 was located predominantly in the eosinophil granules, as shown by both immunogold electron microscopy and a cell fractionation technique that dissociated cell granules from membrane and cytosolic components. IL-4 mRNA colocalized with eosinophils (using sequential immunocytochemistry with an eosinophil-specific (EG2) mAb and in situ hybridization using an IL-4-specific antisense riboprobe) in both cell cytospins from bronchoalveolar lavage fluid from asthmatics as well as skin biopsies obtained from allergen-induced late phase (6-h) reactions in atopic subjects. Using double immunocytochemistry on skin biopsies with eosinophil- and IL-4-specific mAb, 83.5 +/- 3.5% of eosinophils were IL-4+. Conversely, eosinophils accounted for 46.5 +/- 3.9% of the total cells expressing IL-4 immunoreactivity. Thus, human eosinophils express mRNA for IL-4, and the translated product is contained within the crystalloid granule from which it is released after stimulation with serum-coated particles. These observations are consistent with the hypothesis that eosinophils contribute to the development of the Th2 phenotype by T cells infiltrating atopic allergic reactions as well as to IgE synthesis.

A survey of GTP-binding proteins and other potential key regulators of exocytotic secretion in eosinophils. Apparent absence of rab3 and vesicle fusion protein homologues.

We set out to identify potential key regulators of exocytotic fusion in the eosinophil, in the knowledge that granule exocytosis can be stimulated in these cells by intracellular application of nonhydrolyzable analogues of guanosine triphosphate, with Ca2+ acting as a modulator of guanine nucleotide-dependent secretion. To screen for GTP-binding proteins, guinea pig eosinophils were purified from peritoneal washings and subjected to western blotting analysis using specific immune sera raised against recombinant proteins or consensus peptide sequences within proteins of interest. We found a number of heterotrimeric G proteins (G alpha i3, G alpha o, G alpha q11, G alpha s and G beta subunits) and members of the small GTP-binding proteins expressed in eosinophils. Two subtypes of G-protein alpha subunits (G alpha i1 and G alpha z) could not be detected. Separation of subcellular organelles from homogenized eosinophils by density gradient centrifugation revealed that all of the detected GTP-binding proteins were mainly expressed in fractions containing peak plasma membrane and Golgi marker enzyme activities, while G beta subunits were also detected in secretory granule fractions. However, isoforms of Rab3, a putative GTP-binding regulator of exocytotic fusion, were undetectable in eosinophils. Neither, with the exception of syntaxin-3, could we detect any of the proteins belonging to the proposed synaptic vesicle fusion complex (SNAP-25; synaptobrevin (VAMP) and its non-neuronal homologue, cellubrevin; synaptophysin; synaptotagmin). The results from this study, based on western blotting, suggest that eosinophils express a different class of exocytotic fusion complex proteins from those found in neuronal tissues, although a number of potential candidates fulfilling the role of GE were identified in this important inflammatory cell.

Association of granulocyte-macrophage colony-stimulating factor with the crystalloid granules of human eosinophils.

We have previously shown that normal-density human peripheral blood eosinophils transcribe and translate mRNA for granulocyte-macrophage colony-stimulating factor (GM-CSF) and that the intracellular distribution was granular as assessed by light microscopy immunocytochemistry. The present study was conducted to confirm this apparent association between GM-CSF and the crystalloid granule using a subcellular fractionation method for human eosinophils and immunogold electron microscopy (EM). Highly purified (> 99%, by negative selection using anti-CD16 immunomagnetic microbeads) human peripheral blood eosinophils were obtained from four asthmatic subjects (not taking systemic medication), homogenized and density fractionated (5 x 10(7) cells/subject) on linear Nycodenz gradients. Twenty-four fractions were collected from each cell preparation and analyzed for marker enzyme activities as well as total protein. Dot blot analysis with specific monoclonal antibodies (MoAbs) was used to detect the eosinophil granule proteins major basic protein (MBP) and eosinophil cationic protein (ECP). An anti-CD9 MoAb was used as an eosinophil plasma membrane marker. Lactate dehydrogenase (LDH) was used as a cytosolic marker. Immunoreactivity for GM-CSF was detected by a specific enzyme-linked immunosorbent assay using a polyclonal antihuman GM-CSF antibody and confirmed by dot blot. GM-CSF coeluted with the cellular fractions containing granule markers (MBP, ECP, eosinophil peroxidase, hexosaminidase, and arylsulphatase), but not those containing cytoplasm (LDH+) or membrane (CD9+) markers. EM examination of pooled fractions associated with the peak of GM-CSF immunoreactivity confirmed that they contained crystalloid and small granules, but not plasma membrane. In addition, quantification, using immunogold labeling with an anti/GM-CSF MoAb, indicated preferential localization of gold particles over the eosinophil granule cores of intact cells. Thus, our results indicate that GM-CSF resides as a granule-associated, stored mediator in unstimulated human eosinophils.

The structure of the contractile apparatus in ultrarapidly frozen smooth muscle: freeze-fracture, deep-etch, and freeze-substitution studies.

The structure of the smooth muscle contractile apparatus was studied using ultrarapid freezing followed by freeze substitution or by longitudinal freeze-fracture, deep-etch, and platinum-carbon replication. Freeze substitution minimises the detrimental effects of chemical fixation and freeze fracture eliminates them entirely whilst revealing the ultrastructure in three dimensions. Unidirectionally shadowed freeze-fracture replicas of ultrarapidly frozen, relaxed, intact smooth muscle showed a well-preserved actin filament structure the 5.5-nm repeat of the actin subunits was clearly observed. In transversely fractured tissue the thick filaments were revealed, with a distribution comparable to that seen in transverse sections of freeze-substituted muscle. Relaxed muscle permeabilised using Triton X-100 showed a similar structure to that of intact tissue after ultrarapid freezing and examination both by freeze fracture and by freeze examination both by freeze fracture and by freeze substitution; the ratios of actin to myosin were also comparable. In permeabilised, rigorised tissue the structure of the actomyosin complex was revealed in detail; this was especially clear in freeze-substituted muscle. A cross-bridge spacing of 38 nm was measured in freeze-fractured, deep-etched tissue. The structural detail revealed is compatible with a side polar model of the actomyosin interaction and with the sliding filament mechanism of muscle contraction.

Stretch and anesthetic dependency of atrial natriuretic peptide release demonstrated by an ultrastructural assay.

Using an ultrastructural assay developed to quantify the secretion of atrial natriuretic peptide-containing granules, release of the hormone, in response to different degrees of atrial distension, is directly demonstrated at the cellular level. The ultrastructural assay developed uses an in situ tannic acid perfusion technique to arrest the exocytosis of atrial granules in the anesthetized rat. Secretory granules, which retain the capacity to undergo exocytosis throughout a 30-minute tannic acid perfusion, accumulate at the cell surface in a state of fusion with the plasma membrane, with the core contents retained. Quantification of arrested granules thus provides a measure of the rate of granule release and allows the responses to different stimuli to be assessed. By altering the height of the perfusate, perfusion pressure and hence the degree of distension of the right atrium can be increased, and this causes a proportional rise in the release of secretory granules from individual myocytes. An anesthetic regime incorporating fentanyl citrate was found to increase significantly the rate of granule release, and this was further augmented by atrial distension. Quantification of the numbers of cytoplasmic granules under the same conditions did not reveal a reduction in granules. This is thought to be because only a small pool of granules is recruited for exocytosis, and granule production may continue during the perfusion period. Our assay of atrial secretory granule release allows the effect of a variety of stimulatory and inhibitory agents to be assessed directly at the cellular level and provides an independent comparison with previous biochemical data from whole animal and isolated organ studies.

Coated vesicles are implicated in the post-fusion retrieval of the membrane of rat atrial secretory granules.

Using an in situ tannic acid perfusion technique, this study presents evidence that the removal of membrane components from the rat atrial secretory granule membrane after granule exocytosis is mediated by coated vesicles. When tannic acid is used to arrest the post-fusion stages of granule release, coated pit formation occurs on granule membrane, which, although continuous with the sarcolemma, is easily recognised by the membrane omega profile and the continued presence of the granule core. Tannic acid perfusion, before aldehyde fixation, allows a degree of continued cell function, and granule fusions can persist after tannic acid has reached the cell. This results in an increase in the numbers of fusion profiles and the appearance of coated pits on granule membrane at these sites. The proportion of granules with coats increases with perfusion time, suggesting that endocytotic, as well exocytotic events, may be arrested by the action of tannic acid. Coated vesicles are also involved at earlier stages of the release pathway. In other types of secretory system this is considered to represent recycling of membrane proteins as part of the maturation process of the granule. Although arrested granules exhibiting this clathrin coat could have had the coat prior to fusion, as part of the maturation process, our results show that it is more likely to represent a second stage of membrane protein recycling; the postfusion reclamation of proteins from the sarcolemma. This facet of the tannic acid perfusion procedure suggests a general method for quantifying coated pit formation during secretory granule release.

The pathway of atrial natriuretic peptide release--from cell to plasma.

Tannic acid and dextran have been used to arrest the exocytosis of secretory granules in the atria of the rat heart. By immunogold labeling with silver intensification of ultrathin sections, the arrested exocytosing granules are demonstrated to contain atrial natriuretic peptide (ANP). Extracellular core-like structures found in atria treated with tannic acid are also shown to contain atrial natriuretic peptide. This allows a pathway for the release of atrial natriuretic peptide to be traced from the surface of the myocyte, through the endomysium, into the sub-endothelial space and to the abluminal surface of the capillary endothelium. Uptake of atrial natriuretic peptide into the capillary endothelial cells was also detected. Endothelial transport and release of atrial natriuretic peptide appears to involve the caveolae and smooth vesicles of the non-selective endothelial transport system. No labeling was detected in the endothelial cells of the endocardium or in the mesothelial cells of the epicardium. A disruption of arrested granule cores is seen after dextran treatment. This, and the variation in the labeling of extracellular cores observed, is consistent with the possibility that the cleavage of atrial natriuretic peptide prohormone may be initiated upon the fusion of a granule with the plasma membrane, and continue during the passage of the core material through the extracellular space, with a gradual loss of the active moiety from the disrupted core.

Arrested exocytosis of atrial secretory granules.

Release of atrial natriuretic peptide (ANP) from atrial muscle cells is thought to occur by exocytosis of secretory granules, as in other secretory systems. However, in the atrial myocyte, exocytosis has previously proved difficult to detect ultrastructurally. In order to study the mechanism of ANP release and related events, we have applied two separate techniques--tannic acid perfusion and ultrarapid freezing--specifically designed to arrest exocytosis. An increased number of fusion sites was found after ultrathin sectioning and freeze-fracture of tannic acid-treated atria enabling a hypothetical release sequence to be constructed. Detail of granule substructure was also obtained, suggesting the presence of a coat to the secretory granule core. Ultrarapid freezing, followed by freeze-fracture and freeze substitution, has confirmed aspects of the proposed exocytotic sequence, suggesting that this release is not due to the application of tannic acid, and these techniques also produced further evidence for the existence of the granule-core coat. Coated pits and vesicles were also found in large numbers in tannic acid-treated atria and interactions between coated vesicles and secretory granules were visualized. The possible role of coated vesicles in an exocytotic/endocytotic membrane retrieval pathway is discussed.

Rectilinear particle arrays in freeze-fracture replicas of the surface membrane of Paramecium tetraurelia.

Freeze-fracture replicas of the plasma membrane of unfixed, uncryoprotected Paramecium tetraurelia bear large rectilinear arrays of 11 nm particles arranged in 7-11 parallel rows. The arrays are of sufficient size to leave impressions in replicas of the underlying outer alveolar membrane, and are apparent as parallel ridges in replicas of the surface coat of deep-etched cells. By noting the location of arrays in replicas of identified portions of the cortex of P. tetraurelia, it has been possible to map the distribution of arrays over the cell surface. The arrays are found primarily over the anterior surfaces of the cell, covering an area that extends from the preoral suture over the left adoral field and a large portion of the anterior dorsal surface. Freeze-fracture analyses of cells taken from a number of different stages of a culture cycle suggest that the particle arrays are not replicated as an integral part of the cortex during cell division, but are assembled and oriented in the membrane as the cells mature. The appearance of small intramembranous particle complexes in the plasma membrane of cells in logarithmic growth phase supports this hypothesis, possibly representing an assembly stage in the formation of the larger particle arrays. The facts that the particle arrays are apparent in replicas of the surface coat of cells, are found primarily at the anterior of the cell body, and have a highly specific orientation with respect to the cell surface, strongly suggest that they function as chemoreceptors in P. tetraurelia.

Membrane specialisations of the neuromuscular junction of the locust, Schistocerca gregaria.

The ultrastructure of locust (Schistocerca gregaria) neuromuscular junctions as observed in freeze-fracture replicas is described and correlated with their appearance in thin sections of material prepared by various fixation regimes and by use of the sectioned replica technique. Two main specialisations are found at the junction. Postsynaptically there is a concentration of large intramembranous particles partitioning on fracturing with the E-face of the sarcolemma. These are associated with smaller, less defined subparticles thought to be proteinaceous projections seen in thin section. Presynaptically a raised row of particles is present on an elliptical plateau of the P-face of the nerve membrane, corresponding to a densely staining bar present in thin sections prepared using tannic acid and methylamine tungstate. These specialisations are found in both red and white fibres, indicating that the junctions are fast excitatory rather than inhibitory junctions. The bar-shaped particle array is not present 14 days after denervation. These specialisations are compared with those at synapses previously described and their possible function at a glutamatergic neuromuscular junction is discussed.

Membrane specialisation of the extra-junctional sarcolemma of normal and denervated insect muscle.

Corrugated areas of sarcolemma were observed in serial transverse sections. Complexes of intramembranous particles that appear after freeze-fracture replication were concluded to represent the same specialisation. These specialisations, up to 14 microns x 2 microns, are orientated parallel to the long axis of the muscle. Intramembranous particles are concentrated along the peaks of the corrugations, and are associated with the P-face. Corresponding pits are found in the E-face. Fourteen and thirty days after sectioning the excitatory motor-nerve supply to the muscle, corrugated areas 0.5-1 microns x 0.5-1 microns are found. Occurring singly or in groups, their orientation with respect to the long axis of the muscle is more variable than those of control muscles. Thin sections reveal no complementary areas on adjacent fibres or intracellular submembrane attachments or specialisations. A structural role is therefore unlikely. Mitochondria are frequently found in close association with these specialisations. Their possible role as receptors or transmembrane transport systems is discussed.